This application involves a collaboration between two PIs in the Department of Chemistry at the University of Pennsylvania, Professors Dmochowski and Christianson. We are combining our complementary expertise in organic synthesis, xenon-based and fluorescence-based molecular imaging, carbonic anhydrase inhibitor design, and protein X-ray crystallography to develop a new class of xenon magnetic resonance imaging (MRI) agents for early lung cancer detection. Unlike most atomic nuclei, xenon-129 can be hyperpolarized, which produces a ~100,000-fold signal enhancement in a MRI scanner. Furthermore, xenon is very polarizable, which allows it to bind water-soluble organic cages called cryptophanes with micromolar dissociation constants, and the 129Xe magnetic resonance chemical shift is very sensitive to the molecular environment of the cryptophane. These properties motivate the development of xenon biosensors for the detection of cancer biomarkers. Our collaborative studies indicate that hyperpolarized 129Xe nuclear magnetic resonance (NMR) biosensors show tremendous promise for the detection of specific isozymes of the zinc metalloenzyme carbonic anhydrase (CA), as Xe biosensors targeting CA I and II gave distinct resonances with very large chemical shifts and narrow linewidths. We also determined the first crystal structure of a CAII-Xe-biosensor complex, and this structure clearly shows the cryptophane encapsulating a single xenon atom and the benzenesulfonamide moiety coordinating to the active site zinc ion, as designed. We now propose an efficient synthesis for single-enantiomer Xe biosensors, which will greatly facilitate the interpretation of 129Xe NMR spectra, as well as the cocrystallization of these compounds with the CA isozymes. Carbonic anhydase is a validated drug target and cancer biomarker. For example, several CA isozymes, including CA IX and XII, are highly overexpressed in malignant tumors. We have chosen to focus on the development of xenon biosensors for small cell lung cancer (NSCLC) for four reasons: (1) lung cancer is the leading cause of cancer death worldwide, (2) early detection of NSCLC allows treatment with surgical procedures and dramatically improves patient prognosis; (3) CA IX and XII are highly overexpressed in most forms of NSCLC, and (4) hyperpolarized 129Xe is readily delivered to the lungs, where it provides useful spectroscopic signatures. In these studies, we propose to elucidate the full range of CA-cryptophane interactions that produce large 129Xe NMR chemical shifts by determining the structures of multiple CA-Xe biosensor complexes and measuring the hyperpolarized 129Xe NMR spectra for these complexes in solution. Together with Penn Chemistry colleague Jeffery Saven, we will analyze these biophysical data and elaborate computationally designed mutations in CA II that will alter the dipole moment while maintaining protein stability. Computational methods for predicting 129Xe NMR chemical shifts for protein-bound xenon biosensors will also be developed, using CA II as a model system. Using these models, we will then focus on the development of 129Xe NMR biosensors for the early detection of NSCLC, targeting CA IX and XII. Xe biosensors will be developed that give very distinct resonances for CA I, II, IX, and XII, and these will be tested in NSCLC cells via fluorescence microscopy and hyperpolarized 129Xe NMR spectroscopy and imaging. Multiplexing experiments will be performed in lung cancer cells and tissues, using xenon biosensors to identify multiple CA isozymes.